A MICRO-ASPIRATOR CHIP USING VACUUM EXPANDED MICROCHANNELS FOR HIGH-THROUGHPUT MECHANICAL
CHARACTERIZATION OF BIOLOGICAL CELLS W. Kim1 and A. Han1, 2*
1Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA 2Department of Electrical and Computer Engineering, Texas A&M University, College Station, TX, USA
ABSTRACT
Mechanical properties of cells can serve as biomarkers to identify cells depending on their disease states, and thus have the potential to be used in disease diagnostic applications. However, existing techniques are not capable of quanti-fying these properties in statistically significant quantities. To address this, we propose a high-throughput micro-aspirator chip, which can deliver, trap, and deform multiple cells simultaneously at single-cell resolution without skill-dependent micromanipulation. The mechanical property of HeLa cells was measured using the micro-aspirator chip and no dependency of HeLa cell Young’s modulus on the diameter of the cells was found. KEYWORDS: Micropipette aspiration, Cell stiffness, Cell Young’s modulus, Cell mechanics, Single cell analysis
INTRODUCTION
Mechanical properties of living cells have been recently shown to be directly related to the cell types and their condi-tions. Of particular interest is the fact that cancer cells, especially metastatic cancer cells, have vastly different stiffness compared to benign cells. Micropipette aspiration and atomic force microscopy (AFM) are the most commonly used techniques for measuring mechanical properties of single cells [1, 2]. Though powerful and versatile, both traditional glass micropipette aspiration technique and AFM have two drawbacks. First, micromanipulation of glass micropipettes and AFM tips require expertise and extensive operator skills. Second, the serial manipulation process severely limits the throughput. To address these limitations, we have developed a high-throughput micro-aspirator chip that can deliver, trap, and deform multiple cells simultaneously with single-cell resolution without skill-dependent micromanipulation.
The micro-aspirator chip is composed of arrays of 40 cell traps paired with one aspiration channel each through which negative pressure can be applied to aspirate the trapped cell. The principle of cell trapping is based on flow resis-tance inside the microfluidic channel (Figure 1) [3]. Once the first trap is filled with a cell, the next cell coming in passes by the trap and is captured in the next trap. After all traps are filled with cells, negative pressure can be applied to the 40 aspiration channels simultaneously using hydrostatic pressure. One of the key design concept of this device is that the aspiration channel is positioned at the center of the cell both in vertical and horizontal direction to obtain a good seal be-tween the cell and the aspiration channel, just like a traditional micropipette (Figure 2). Devices have been reported pre-viously to make raised aspiration channels for planar patch-clamping devices [4, 5], but not for cell stiffness analyses.
Figure 1: Schematic diagram showing the cell trapping and cell aspiration principle. (1, 2) When the trap is empty, flow resistance along the straight channel is lower than that of the loop channel, and the main flow stream goes through the cell trap. (3) Once a cell is trapped, the main flow stream goes through the loop channel, carrying the second cell to the next trap. (4) Negative pressure is applied to aspirate the trapped cell.
Figure 2: (a) Cross-sectional view of a cell undergoing aspiration into a microfluidic channel. (b) Cell aspiration into a raised microfluidic channel.
FABRICATION The PDMS device having raised aspiration channels was fabricated by utilizing the PDMS membrane vacuum expan-
sion method (Figure 3) [5]. First, the microfluidic channel layer was replicated from a double-layer SU-8 master mold and bonded to a 2 µm-thick PDMS membrane. PDMS pre-mixture was then poured on top of the membrane. Vacuum was applied to the device during the PDMS polymerization process. Pressure difference between inside and outside of the enclosed microfluidic channel caused the expansion of the membrane, mainly on the cell traps and the cell delivery channels. Due to the narrow width (3 µm), the membrane on the aspiration channel was not expanded. Figure 4 shows the PDMS membrane thickness optimization process. Through process optimization, this vacuum expansion process took only 12 minutes, significantly reducing the previously reported 24-hour processing time [5].
Figure 3: Schematic of the fabrication steps. (a) PDMS block made out of a double-layer SU-8 master. (b) PDMS mem-brane bonded on the microfluidic layer. (c) Vacuum expanded microfluidic channels.
Figure 4: Vacuum expansion process optimization. Microscopic images of cross sections of the aspiration channel (1) and the expanded cell delivery channel (2). (a) PDMS membrane thickness: 2 µm, sufficient expansion. (b) PDMS mem-brane thickness: 7 µm, insufficient expansion. (c) PDMS membrane thickness: 1 µm, collapsed membrane attached to the bottom of the channel. EXPERIMENTAL
The cell trapping and aspiration capability of the micro-aspirator chip was tested with HeLa cells. HeLa cells suspended in culture media (1 x 106 cells/ml ) was injected into the inlet of the cell delivery channel using a syringe. Following cell trapping, 6 incremental steps of suction pressure between 1-6 kPa was applied through the aspiration channel using hydrostatic pressure generated by a height-adjustable PBS reservoir (Figure 5) and the aspiration length was measured (Figure 6 (b)). A pressure transducer was used to accurately measure the amount of pressure applied. All processes were observed through an inverted microscope and re-corded using a CCD camera.
Figure 5: Schematic diagram of the experimental setup.
RESULTS AND DISCUSSION As shown in Figure 6 (a), HeLa cells were sequentially captured at each trap. The cell trapping efficiency was almost
100%. Using Theret et al.’s model [6] that shows the relationship between the cell aspiration length and the cell’s Young’s modulus, HeLa cell Young’s modulus of 1.3 ± 0.8 kPa (n = 54) was obtained using three devices (Figure 7 (a)). Device-to-device variation of the measured value was less than 15% (n = 3), showing good repeatability. No dependency of the Young’s modulus on the cell diameters was found (Figure 7 (b)). However, the measured Young’s modulus was relatively low compared to previously reported values. This might be due to the square geometry of the aspiration chan-nel resulting in deviation from the true Young’s modulus, as well as from the variation in cell conditions and experimen-tal procedures in the various reports. Repeated studies with larger number of cells are currently being conducted to fur-ther analyze the result.
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Figure 6: (a) Microscopic image of cells captured inside arrays of cell traps. White arrow heads indicate the trapped cells. (b) A single HeLa cell aspirated into the aspiration channel at various negative pressures.
Figure 7: (a) Suction pressure vs. average aspiration length of HeLa cells. (b) Correlation between the Young’s mod-ulus and the cell diameter. No significant correlation was observed, suggesting that the size of cells did not influence the stiffness. CONCLUSION
A simple and easy to use micro-aspirator chip that can deliver, trap, and deform 20 cells simultaneously at single-cell resolution has been demonstrated. The developed system eliminated the labor-intensive nature of the conventional mi-cropipette aspiration, enabled fast and accurate characterization of single cells through cell trapping using flow resistance inside microchannel and cell deforming using hydrostatic pressure, and achieved high-throughput measurement of cell’s Young’s modulus. By realizing the conventional micropipette aspiration method into a chip-based system, the developed micro-aspirator chip is expected to provide an in-depth look into mechanical deformation occurring at single-cell level and pathological status of single cells with high-throughput. The device can not only be used for various single cell stud-ies but also for disease diagnosis such as cancer detection. REFERENCES [1] R. Hochmuth , “Micropipette aspiration of living cells,” Journal of Biomechanics, vol. 33, pp. 15-22, (2000). [2] S.E. Cross et al., “Nanomechanical analysis of cells from cancer patients,” Nature, vol. 2, pp. 780-783, (2007). [3] W. Tan and S. Takeuchi, “A trap-and-release integrated microfluidic system for dynamic microarray applications,”
PNAS, vol. 104, pp. 1146-1151, (2007). [4] A.Y. Lau, P.J. Hung, A.R. Wu and L.P. Lee, “Open-access microfluidic patch-clamp array with raised lateral cell
trapping sites,” Lab Chip, vol. 6, pp. 1510-1515, (2006). [5] J. Seo and L.P. Lee, “Self-raised circular orifices for lateral patch-clamping array chips,” Proc. Micro Total Analy-
sis Systems 2005, pp. 1215-1217, (2005). [6] D.P. Theret et al., “The application of a homogeneous half-space model in the analysis of endothelial cell micropi-
pette measurement,” Journal of Biomedical Engineering, vol. 110, pp. 190-199, (1988). CONTACT *Arum Han, tel: +1- 979-845-9686; [email protected]
